Evaluation of immunoassay methods for the screening of cocaine metabolites in urine

Immunoassay kits for urine cocaine (and metabolite) screening, obtained from two commercial sources, were examined for correlation of their results, expressed in terms of equivalent benzoylecgonine concentration, with the gas chromatography/mass spectrometry (GC/MS) concentration of benzoylecgonine. The correlation coefficients obtained, based on 62 (out of a total sample population of 3295) highly relevant samples, were 0.467 and 0.766 for Abuscreen (ARIA) and TDx (TDX), respectively. The preliminary screen cutoff values, which correspond to 150 ng/mL benzoylecgonine (as determined by GC/MS), were calculated based on the resulting regression equations and found to be 380 and 190 ng/mL for ARIA and TDX, respectively. With these cutoff values, ARIA generates 5 false negatives and 16 unconfirmed presumptive positives, while TDX results in 3 false negatives and 6 unconfirmed presumptive positives.     

Confirmation of Syva enzyme multiple immunoassay technique (EMIT) d.a.u. and Roche Abuscreen radioimmunoassay (RIA) (125I) urine cannabinoid immunoassays by gas chromatographic/mass spectrometric (GC/MS) and bonded-phase adsorption/thin-layer chromatographic (BPA-TLC) methods

Thirty human urines screened positive by the Syva enzyme multiple immunoassay technique (EMIT) d.a.u. urine cannabinoid assay were also positive for the major marijuana urinary metabolite 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) when assayed by gas chromatographic/mass spectrometric (GC/MS) and a noninstrumental qualitative bonded-phase adsorption/thin-layer chromatographic (BPA-TLC) technique. The noninstrumental BPA-TLC procedure was the simpler of the two techniques to perform and interpret. Assay of these same samples by the Roche Abuscreen radioimmunoassay (RIA) for cannabinoids (125I) revealed that reliance on the 100-ng/mL equivalent positive calibrator yielded a high incidence of false negative results (10 out of 30). The performance of these same 4 assays on 30 true negatives also was evaluated. All samples were negative for cannabinoids by EMIT and RIA, and for THC-COOH by BPA-TLC. GC/MS assay, however, detected spurious low levels of approximately 5-ng/mL THC-COOH in two instances. Because of this, a reliability level of 10 ng/mL was set for the routine quantitative confirmation of THC-COOH by the GC/MS method.     

Urinary excretion profiles of 11-nor-9-carboxy-Delta9-tetrahydrocannabinol. Study III. A Delta9-THC-COOH to creatinine ratio study

Huestis and Cone reported in [J. Anal. Toxicol. 22 (1998) 445] that serial monitoring of Delta9-THC-COOH/creatinine ratios in paired urine specimens collected at least 24h apart could differentiate new drug use from residual Delta(9)-THC-COOH excretion following acute marijuana use in a controlled setting. The best accuracy (85.4%) for predicting new marijuana use was for a Delta(9)-THC-COOH/creatinine ratio > or = 0.5 (dividing the Delta9-THC-COOH/creatinine ratio of specimen no. 2 by the specimen no. 1 ratio). In previous studies in this laboratory [J. Anal. Toxicol. 23 (1999) 531 and Forensic Sci. Int. 133 (2003) 26], urine specimens were collected from chronic marijuana users > or = 24 h or > = 48 h apart in an uncontrolled setting. Subjects with a history of chronic marijuana use were screened for cannabinoids with the EMIT II Plus cannabinoids assay (cut-off 50 ng/ml) followed by confirmation for Delta9-THC-COOH by GC-MS (cut-off 15 ng/ml). Creatinine was analyzed as an index of dilution. The objective of the present study was to evaluate whether creatinine corrected specimens could differentiate new marijuana or hashish use from the excretion of residual Delta(9)-THC-COOH in chronic marijuana users based on the Huestis 0.5 ratio. Urine specimens (N=376) were collected from 29 individuals > or = 96 h between urine collections. The mean urinary Delta9-THC-COOH concentration was 464.4 ng/ml, mean Delta9-THC-COOH/creatinine ratio (ng/(ml Delta9-THC-COOH mmoll creatinine)) was 36.8 and the overall mean Delta9-THC-COOH/creatinine ratio of specimen 2/mean Delta9-THC-COOH/creatinine ratio of specimen 1 was 1.37. The Huestis ratio calculation indicated new drug use in 83% of all sequentially paired urine specimens. The data were sub-divided into three groups (Groups A-C) based on mean Delta9-THC-COOH/creatinine values. Interindividual mean Delta9-THC-COOH/creatinine values ranged from 4.7 to 13.4 in Group A where 80% of paired specimens indicated new drug use (N=10) and 20.4-39.6 in Group B where 83.6% of paired specimens indicated new drug use (N=7). Individual mean Delta9-THC-COOH/creatinine values ranged from 44.2 to 120.2 in Group C where 84.5% of paired urine specimens indicated new marijuana use (N=12). Correcting Delta9-THC-COOH excretion for urinary dilution and comparing Delta9-THC-COOH/creatinine concentration ratios of sequentially paired specimens (collected > or = 96 h apart) may provide an objective indicator of ongoing marijuana or hashish use in this population.     

Urinary excretion profiles of 11-nor-9-carboxy-Delta9-tetrahydrocannabinol: a Delta9-THC-COOH to creatinine ratio study #2

Subjects with a history of chronic marijuana use were screened for cannabinoids in urine specimens with the EMIT((R)) II Plus cannabinoids assay with a cut-off value of 50 ng/ml. All presumptively positive specimens were submitted for confirmatory analysis for the major urinary cannabinoid metabolite (Delta(9)-THC-COOH) by GC-MS with a cut-off value of 15 ng/ml. Creatinine was analyzed in each specimen as an index of dilution. Huestis and Cone [J. Anal. Toxicol. 22 (1998) 445] reported that serial monitoring of Delta(9)-THC-COOH to creatinine ratios in paired urine specimens collected at least 24h apart could differentiate new drug use from residual Delta(9)-THC-COOH excretion. The best accuracy (85.4%) for predicting new marijuana use was a Delta(9)-THC-COOH/creatinine ratio > or =0.5 (dividing the Delta(9)-THC-COOH to creatinine ratio of specimen 2 by the specimen 1 ratio). In a previous study in this laboratory [J. Anal. Toxicol. 23 (1999) 531], urine specimens were collected from chronic marijuana users at least 24h apart and dilute urine specimens (creatinine values <2.2 micromol/l) were excluded from the data analysis. The objective of the present study was to determine whether creatinine corrected urine specimens positive for cannabinoids could differentiate new marijuana use from the excretion of residual Delta(9)-THC-COOH in chronic users of marijuana based on the Huestis 0.5 ratio. Urine specimens (N=946) were collected from 37 individuals with at least 48h between collections. All urine specimens were included in the data review irrespective of creatinine concentration. The mean urinary Delta(9)-THC-COOH concentration was 302.4 ng/ml, mean Delta(9)-THC-COOH/creatinine ratio (ng/ml Delta(9)-THC-COOH/(mmol/l) creatinine) was 29.3 and the Huestis ratio calculation indicated new drug use in 83% of all sequentially paired urine specimens. The data were sub-divided into three groups (A-C) based on the mean Delta(9)-THC-COOH/creatinine values. Interindividual Delta(9)-THC-COOH/creatinine mean values ranged from 2.2 to 13.8 in group A (264 specimens, N=15 subjects) where 80.7% of paired specimens indicated new drug use. In group B, mean Delta(9)-THC-COOH/creatinine values ranged from 15.3 to 37.8 in 444 specimens (N=14 subjects) and 83.3% of paired specimens indicated new drug use. In group C, individual mean Delta(9)-THC-COOH/creatinine values were >40.1 (41.3-132.5) in 238 urine specimens (N=8 subjects) and 85.3% of paired urine specimens indicated new marijuana use. Correcting Delta(9)-THC-COOH excretion for urinary dilution and comparing Delta(9)-THC-COOH/creatinine concentration ratios of sequentially paired specimens (collected at least 48h apart) provided an objective indicator of new marijuana use in this population.     

Urinary excretion of benzoylecgonine following ingestion of Health Inca Tea

Four males ingested one cup of Health Inca Tea which contained 1.87 mg of cocaine. Urine specimens collected for 36 h post-ingestion were analyzed for benzoylecgonine (BE) by EMIT-d.a.u., TDx and gas chromatography/mass spectrometry (GC/MS). Positive immunoassay results were obtained for 21-26 h post tea ingestion. Discrepant immunoassay results occurred with only one specimen: EMIT positive; TDx negative, 0.25 mg/l; GC/MS, 0.273 mg/l. Quantitative TDx results were well correlated with GC/MS results, r2 = 0.963, n = 45. Maximum urinary BE concentrations ranged from 1.4-2.8 mg/l, occurring from 4-11 h, post ingestion. Total BE excretion in 36 h ranged from 1.05 to 1.45 mg, 59-90% of the ingested cocaine dose. Urinary excretion rate constant (Km) ranged from -0.073 to 0.111/h. Health Inca Tea ingestion should be considered when interpreting urinary BE concentrations.     

Validity testing of the TDx Cocaine Metabolite Assay with human specimens obtained after intravenous cocaine administration

Clinical specimens obtained from human subjects after intravenous cocaine administration were analyzed by the TDx Cocaine Metabolite Assay (TDx) and by GC/MS for benzoylecgonine. The TDx results were significantly correlated with results by GC/MS assay with no evidence of bias in the TDx assay. All cocaine metabolite positive specimens (greater than or equal to 300 ng/ml) were confirmed by GC/MS. Detection times to the last positive specimen by TDx assay and GC/MS assay of four subjects after a 20-mg intravenous dose of cocaine ranged from 29.3 to 39.1 h and 27.9 and 36.6 h, respectively. Overall, the TDx assay was found to be highly specific and accurate for the detection and measurement of benzoylecgonine in urine.     

Validity testing of commercial urine cocaine metabolite assays: I. Assay detection times, individual excretion patterns, and kinetics after cocaine administration to humans

A validity study of eight commercial urine assays for detection of cocaine metabolite was performed on clinical specimens collected from human subjects who received single 20-mg intravenous doses of cocaine hydrochloride. The specimens were collected under controlled conditions and analyzed in random order under blind conditions. Benzoylecgonine concentration in each specimen also was determined by gas chromatography/mass spectrometry (GC/MS). Mean times of detection of the last positive specimen (greater than or equal to 300 ng/mL of benzoylecgonine equivalents) after cocaine administration varied among seven of the commercial tests from 16.9 to 52.9 h in the following ascending order: Toxi-Lab less than TDx = EMIT dau = EMIT st less than Abuscreen less than Coat-A-Count = Double Antibody. In contrast, a commercial spot test (KDI Quik Test) which was evaluated for detection of cocaine metabolite produced both false positives and false negatives for benzoylecgonine and was not considered to be a valid test for detection of cocaine metabolite. Half-lives of excretion of benzoylecgonine among four subjects varied from 5.9 to 7.9 h, and overall recovery of benzoylecgonine varied from 15.0 to 34.3% of the administered dose of cocaine.     

Urinary excretion profiles of 11-nor-9-carboxy-delta9-tetrahydrocannabinol and 11-hydroxy-delta9-THC: cannabinoid metabolites to creatinine ratio study IV

The objective of this study was to compare urinary excretion patterns of two cannabinoid metabolites in subjects with a history of chronic marijuana use. The first metabolite analyzed was nor-9-carboxy-delta9-tetrahydrocannabinol (delta9-THC-COOH), the major urinary cannabinoid metabolite that is pharmacologically inactive. The second metabolite 11-OH-delta9-THC is an active cannabinoid metabolite and is not routinely measured. Urine specimens were collected from four subjects on 12-20 occasions > or = 96 h apart in an uncontrolled clinical setting. Creatinine was analyzed in each urine specimen by the colorimetric modified Jaffé reaction on a SYVA 30R biochemical analyzer. All urine specimens analyzed for 11-OH-delta9-THC had screened positive for cannabinoids with the EMIT II Plus cannabinoids assay (cut-off 50 ng/mL) on a SYVA 30R analyzer and submitted for delta9-THC-COOH confirmation by GC-MS (cut-off concentration 15 ng/mL). Eleven-OH-delta9-THC was measured by GC-MS with a cut-off concentration of 3 ng/mL. Both GC-MS methods for cannabinoid metabolites used deuterated internal standards for quantitative analysis. The mean (range) of urinary delta9-THC-COOH concentration was 1153 ng/mL (78.7-2634) with a cut-off of 15 ng/mL. The mean (range) of delta9-THC-COOH/creatinine ratios (ng/mL delta9-THC-COOH/mmol/L creatinine) was 84.1 (8.1-122.1). The mean (range) urinary of 11-OH-delta9-THC concentration was 387.6 ng/mL (11.9-783) with a cut-off of 3 ng/mL, and the mean (range) of 11-OH-delta9-THC/creatinine ratio (ng/mL 11-OH-delta9-THC/mmol/L creatinine) was 29.7 (1.2-40.7). Of the 63 urine specimens submitted for delta9-THC-COOH confirmation by GC-MS, 59/63 urine specimens (94%) were positive for delta9 -THC-COOH and 51/63 (81%) were positive for 11-OH-delta9-THC. Overall, the concentrations of 11-OH-delta9-THC in urine specimens collected > or = 96 h apart were lower than delta9-THC-COOH concentrations in 50/51 of the urine specimens in this population. Further urinary cannabinoid excretion studies are needed to assess whether 11-OH-delta9-THC analyses have a role when assessing previous marijuana or hashish use in chronic users whose urine specimens remain positive for delta9-THC-COOH for an extended period of time after last drug use.     

Hair analysis for delta(9)-THC,delta(9)-THC-COOH,CBN and CBD,by GC/MS-EI. Comparison with GC/MS-NCI for delta(9)-THC-COOH

A sensitive analytical method was developed for quantitative analysis of delta(9)-tetrahydrocannabinol (delta(9)-THC), 11-nor-delta(9)-tetrahydrocannabinol-carboxylic acid (delta(9)-THC-COOH), cannabinol (CBN) and cannabidiol (CBD) in human hair. The identification of delta(9)-THC-COOH in hair would document Cannabis use more effectively than the detection of parent drug (delta(9)-THC) which might have come from environmental exposure. Ketamine was added to hair samples as internal standard for CBN and CBD. Ketoprofen was added to hair samples as internal standard for the other compounds. Samples were hydrolyzed with beta-glucuronidase/arylsulfatase for 2h at 40 degrees C. After cooling, samples were extracted with a liquid-liquid extraction procedure (with chloroform/isopropyl alcohol, after alkalinization, and n-hexane/ethyl acetate, after acidification), which was developed in our laboratory. The extracts were analysed before and after derivatization with pentafluoropropionic anhydride (PFPA) and pentafluoropropanol (PFPOH) using a Hewlett Packard gas chromatographer/mass spectrometer detector, in electron impact mode (GC/MS-EI). Derivatized delta(9)-THC-COOH was also analysed using a Hewlett Packard gas chromatographer/mass spectrometer detector, in negative ion chemical ionization mode (GC/MS-NCI) using methane as the reagent gas. Responses were linear ranging from 0.10 to 5.00 ng/mg hair for delta(9)-THC and CBN, 0.10-10.00 ng/mg hair for CBD, 0.01-5.00 ng/mg for delta(9)-THC-COOH (r(2)>0.99). The intra-assay precisions ranged from <0.01 to 12.40%. Extraction recoveries ranged from 80.9 to 104.0% for delta(9)-THC, 85.9-100.0% for delta(9)-THC-COOH, 76.7-95.8% for CBN and 71.0-94.0% for CBD. The analytical method was applied to 87 human hair samples, obtained from individuals who testified in court of having committed drug related crimes. Quantification of delta(9)-THC-COOH using GC/MS-NCI was found to be more convenient than GC/MS-EI. The latter may give rise to false negatives due to the detection limit.     

Comparison of the MTP immunoassay with EMIT in blood screening for drugs

We compared the MTP immunoassay with EMIT for the screening of drugs of abuse (opiates, cannabinoids, cocaine metabolites and amphetamines) in whole blood samples. These blood samples were obtained from the German police, when driving under the influence of drugs of abuse was suspected. For screening with the MTP immunoassay 25 microliters of serum or blood (without any pretreatment) was pipetted into the wells of the microtiter plates and the procedure was followed as described. Prior to screening with a Cobas Mira and EMIT reagents, the samples were treated with acetone to precipitate serum proteins. The cutoff for all drugs of abuse was set at 10 ng per ml of serum or blood. In most cases there was a good agreement between the negative and positive results of the two screening assays. The agreement between the two assays in the detection of opiates and cocaine was 91% and 93%, respectively, and for cannabinoids and amphetamines approximately 80%. The MTP immunoassay was more sensitive than EMIT for the detection of cannabinoids--but at the same time the MTP immunoassay was less specific. Both screening assays have a sensitivity of 100% for the detection of opiates and cocaine, but the specificity of the EMIT--also for opiates--was substantially lower. The MTP immunoassay has in respect to amphetamines a very high sensitivity, whereas the sensitivity of EMIT for amphetamines is inacceptable due to losses during sample preparation. The specificity of MTP immunoassay for amphetamines is not optimal, because a relatively large amount of samples tested false-positive for amphetamines at the cutoff of 10 ng/ml. In summary the MTP immunoassay, although not automated, performs well in comparison with EMIT, especially if the sample preparation for EMIT testing ist considered.     

Fast confirmation of 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH) in urine by LC/MS/MS using negative atmospheric-pressure chemical ionisation (APCI)

A fast method using automated solid-phase extraction (SPE) and short-column liquid-chromatography coupled to tandem mass-spectrometry (LC/MS/MS) with negative atmospheric-pressure chemical ionisation (APCI) has been developed for the confirmation of 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH) in urine samples. This highly specific method which combines chromatographic separation and MS/MS-analysis can be used for the confirmation of positive immunoassay results with a NIDA cut-off of 15ng/ml. The conjugates of THC-COOH were hydrolysed prior to SPE, and a standard SPE was performed using C18-SPE columns. No derivatisation of the extracts was needed as in GC/MS analysis, and the LC run-time was 6.5min by gradient elution with a retention time of 2.4min. Linearity of calibration was obtained in the range between 0 and 500ng/ml (correlation coefficient R(2)=0.998). Using linear regression (0-50ng/ml) the limit of detection (LOD) was 2.0ng/ml and the limit of quantitation (LOQ) was 5.1ng/ml; day-to-day reproducibility and precision were tested at 15 and 250ng/ml and were 13.4ng/ml+/-3.3% and 255.8ng/ml+/-4.5%, respectively.     

Comparison of Abbott fluorescence polarization immunoassay (FPIA) and Roche radioimmunoassay for the analyses of cannabinoids in urine specimens.

Abbott fluorescence polarization immunoassay (FPIA) and Roche Abuscreen radioimmunoassay (RIA) were compared qualitatively with 142 urine specimens containing 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid. Similar qualitative results were obtained in 132 specimens. When discrepent results were observed, all negative results were within 20% of the 100 ng/mL cut-off. We concluded that FPIA and RIA give comparable results to each other.     

The determination of drugs of abuse in whole blood by means of FPIA and EMIT-dau immunoassays--a comparative study

Six groups of common drugs of abuse (cannabinoids, benzoylecgonine, opiates, barbiturates, benzodiazepines and amphetamines) were determined in whole blood after acetone precipitation, using enzyme multiplied immunoassay (EMIT dau) and fluorescence polarisation immunoassay (FPIA--Abbott TDx and ADx) methods. Both methods, designed primarily for urine, allowed the determination of all above mentioned class of drugs but amphetamine. Only 1 ml of a pre- or postmortem blood sample was needed. The sensitivity of cannabinoids determination was higher by FPIA. The FPIA method gave more precise results, particularly in the case of autopsy blood. The method was applied for drug screening in autopsy and police blood samples. The results (both positive and negative) were in agreement with those obtained with chromatographic methods.     

Screening for cannabinoids in blood using EMIT: concentrations of delta-9-tetrahydrocannabinol in relation to EMIT results.

The EMIT d.a.u. cannabinoid assay of methanolic extracts of blood was found to be usable as a screening method in cases of suspected impairment by cannabis, when delta-9-tetrahydrocannabinol (THC) was analysed in the subsequent assay. A prerequisite is that the blood sample is taken some time after cannabis smoking. When a cut-off limit corresponding to 50 nM delta-9-tetrahydrocannabinol carboxylic acid (17 ng/ml) was used, 86% of the EMIT positive blood samples contained THC concentrations above the cut-off limit of 1 nM (0.3 ng/ml). A high EMIT result gave a high probability of finding a high THC concentration in the subsequent confirmation analysis.     

Studies on the stability and detection of cocaine, benzoylecgonine, and 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid in whole blood using Abuscreen radioimmunoassay

A study was undertaken to assess the stability and the radioimmunoassay (RIA) detection of cocaine, benzoylecgonine (BZE), and 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in whole blood while stored in 4 different kinds of blood collection tubes for up to 30 days at refrigeration and room temperatures. At various intervals, the tubes were sampled and analyzed using Abuscreen RIA. Also, semi-quantitative data derived from RIA analysis of forensic blood specimens were compared with quantitative data acquired using gas chromatography (GC) or GC/mass spectrometry (GC/MS) on the same specimens. RIA and chromatographic studies revealed that BZE and THC-COOH were stable in blood under all conditions studied. Cocaine, however, was found not to be stable in blood, especially when stored at room temperatures. Despite cocaine's instability in blood, RIA was able to detect the presence of cocaine and its breakdown products in blood under all conditions studied.     

Analysis of 11-nor-9-carboxy-delta(9)-tetrahydrocannabinol in biological samples by gas chromatography tandem mass spectrometry (GC/MS-MS)

Gas chromatography tandem mass spectrometry (GC/MS-MS) analysis of 11-nor-carboxy-delta(9)-tetrahydrocannabinol (delta(9)-THC-COOH), the major metabolite of delta(9)-tetrahydrocannabinol, in biological samples is reported. The proposed method, using deuterated delta(9)-THC-COOH as an internal standard, is able to detect the major metabolite of cannabis derivatives at very low levels (picograms/millilitre) with high specificity. These characteristics render the proposed analytical procedure suitable for confirmatory analysis in drug testing for cannabis use.     

Optimization and validation of CEDIA drugs of abuse immunoassay tests in serum on Hitachi 912

Due to sensitive limits of detection of chromatographic methods and low limit values regarding the screening of drugs under the terms of impairment in safe driving (§ 24a StVG, Street Traffic Law in Germany), preliminary immunoassay (IA) tests should be able to detect also low concentrations of legal and illegal drugs in serum in forensic cases. False-negatives should be avoided, the rate of false-positive samples should be low due to cost and time. An optimization of IA cutoff values and a validation of the assay is required for each laboratory. In a retrospective study results for serum samples containing amphetamine, methylenedioxy derivatives, cannabinoids, benzodiazepines, cocaine (metabolites), methadone and opiates obtained with CEDIA drugs of abuse reagents on a Hitachi 912 autoanalyzer were compared with quantitative results of chromatographic methods (gas or liquid chromatography coupled with mass spectrometry (GC/MS or LC/MS)). Firstly sensitivity, specificity, positive and negative predictive values and overall misclassification rates were evaluated by contingency tables and compared to ROC-analyses and Youden-Indices. Secondly ideal cutoffs were statistically calculated on the basis of sensitivity and specificity as decisive statistical criteria with focus on a high sensitivity (low rates of false-negatives), i.e. using the Youden-Index. Immunoassay (IA) and confirmatory results were available for 3014 blood samples. Sensitivity was 90% or more for nearly all analytes: amphetamines (IA cutoff 9.5 ng/ml), methylenedioxy derivatives (IA cutoff 5.5 ng/ml), cannabinoids (IA cutoff 14.5 ng/ml), benzodiazepines (IA cutoff >0 ng/ml). Test of opiates showed a sensitivity of 86% for a IA cutoff value of >0 ng/ml. Values for specificity ranged between 33% (methadone, IA cutoff 10 ng/ml) and 90% (cocaine, IA cutoff 20 ng/ml). Lower cutoff values as recommended by ROC analyses were chosen for most tests to decrease the rate of false-negatives. Analyses enabled the definition of cutoff values with good values for sensitivity. Small rates of false-positives can be accepted in forensic cases.     

Time of drug elimination in chronic drug abusers. Case study of 52 patients in a "low-step" detoxification ward

The elimination time of illicit drugs and their metabolites is of both clinical and forensic interest. In order to determine the elimination time for various drugs and their metabolites we recruited 52 volunteers in a protected, low-step detoxification program. Blood samples were taken from each volunteer for the first 7 days, daily, urine sample for the first 3 weeks, daily. Urine was analyzed using a fluorescence-polarization immunoassay (FPIA) and gas chromatography/mass spectrometry (GC/MS), serum using GC/MS. The elimination times of the drugs and/or their metabolites in urine and serum as well as the tolerance intervals/confidence intervals were determined. Due to the sometimes extremely high initial concentrations and low cut-off values, a few of the volunteers had markedly longer elimination times than those described in the literature. The cut-off values were as follows: barbiturates II (200ng/ml), cannabinoids (20ng/ml), cocaine metabolites (300ng/ml), opiates (200ng/ml). GC/MS detected the following maximum elimination times: total morphine in urine up to 270.3h, total morphine and free morphine in serum up to 121.3h, monoacetylmorphine in urine up to 34.5h, 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THC-COOH) in urine up to 433.5h, THC-COOH in serum up to 74.3h, total codeine in urine up to 123h, free codeine in urine up to 97.5h, total codeine in serum up to 29h, free codeine in serum up to 6.3h, total dihydrocodeine (DHC) in urine up to 314.8h, free DHC in urine up to 273.3h, total and free DHC in serum up to 50.1h. Cocaine and its metabolites were largely undetectable in the present study.     

Detection of drugs in saliva of impaired drivers

This study examined the feasibility of detecting drugs using saliva samples obtained from impaired drivers. Screening procedures on 1- to 2-mL samples were for cannabinoids, volatiles, benzodiazepines, and other acidic/neutral/basic drugs. Methodology consisted of enzyme multiple immunoassay technique (EMIT) and temperature programmed gas chromatography with confirmation by gas chromatography/mass spectrometry (GC/MS). Fifty-six samples were obtained from drivers arrested for suspicion of impaired driving. Other than alcohol, the major drugs detected were cannabinoids and diazepam. Cocaine was found in one case.     

Validity testing of commercial urine cocaine metabolite assays: IV. Evaluation of the EMIT d.a.u. cocaine metabolite assay in a quantitative mode for detection of cocaine metabolite

The EMIT d.a.u. cocaine metabolite assay (EMIT dau) was evaluated in a quantitative mode for analysis of clinical specimens obtained after controlled cocaine administration to human subjects. The quantitative results showed high concordance with those of gas chromatography/mass spectrometry (GC/MS) assays of the same specimens for benzoylecgonine, and no false positive or false negative results were obtained. The evaluation also included analysis of standardized solutions containing benzoylecgonine, cocaine, and other cocaine metabolites and isomers. The EMIT dau antibody demonstrated high selectivity for benzoylecgonine. The precision was somewhat less than that reported earlier for other commercial cocaine metabolite immunoassays. Quantitation of initial screening results from EMIT dau testing can serve as a useful guide for confirmation by GC/MS in forensic science urine testing.